Immobilization of biomolecules on substrates by attaching them to adsorbed bridging biomolecules

ABSTRACT

An assay article for detection first biomolecules contained in a sample is described. The assay article includes a substrate having a modified surface and a first biomolecule directly adsorbed and immobilized on the modified surface of the substrate without linking moieties. A second biomolecule is bound to or adsorbed on the first biomolecule. Also disclosed is a method of making the assay article. A first biomolecule (other than an adhesive protein) is contacted with a modified surface of a substrate. The substrate is dried to directly adsorb the first biomolecule and immobilize it on the modified surface of the substrate without additional fixing steps to form an activated substrate. Then, a second biomolecule is contacted with the activated substrate under conditions sufficient for the first biomolecule to bind the second biomolecule.

[0001] This application is a continuation-in-part of application Ser. No. 09/694,701, filed on Oct. 23, 2000.

AREA OF THE ART

[0002] The present invention relates generally to solid substrates with immobilized biomolecules. In particular, the invention relates to assay articles with adsorbed first biomolecules to which second biomolecules are bound. The invention also relates to methods of construction of such assay articles and methods of their use in detection of target biomolecules.

DESCRIPTION OF THE PRIOR ART

[0003] Analysis of unknown biomolecular targets often involves their specific binding to known biomolecular probes. The most common technique employing immobilized biomolecules is the Southern blot hybridization technique, in which a set of DNA targets is immobilized on a membrane and a solution containing labeled DNA probe molecules is used to bathe the membrane under conditions where complementary molecules will anneal. In an analogous technique called Northern blot hybridization, RNA targets are immobilized on membranes and anneal to complementary RNA probes. Reverse blot hybridization employs the opposite approach. Instead of immobilizing DNA targets, a set of DNA probes is immobilized on a solid surface and the unknown labeled DNA target is present in the liquid phase.

[0004] Arrays, constructed by attaching a plurality of the same or different biomolecules to discrete isolated areas on the surface of the substrate, are becoming increasingly important tools in the analysis of unknown biomolecules, such as gene expression analysis, DNA sequencing, mutation detection, polymorphism screening, linkage analysis, genotyping single nucleotide polymorphisms (SNPs), and screening for alternative splice variants in gene transcripts.

[0005] Gene expression analysis is a method of critical importance to basic molecular biological research. Since, in higher organisms, the choice of genes being expressed in any given cell has a profound effect on the nature of the cell, gene expression analysis can provide a key to the diagnosis, prognosis, and treatment of a variety of diseases in animals, including humans and plants. Additionally, gene expression analysis can be used to identify differentially-expressed novel genes, to correlate a gene expression to a particular phenotype, to screen for a disease predisposition, and to conduct toxicity testing.

[0006] Typically, in the gene expression analysis, an array of probe nucleic acids is formed by attaching a set of individual gene-specific probes to a solid substrate in a regular pattern, so that the location of each probe is known. The array is contacted with a sample containing target nucleic acids under hybridization conditions. The hybrids are detected using a wide variety of methods, most commonly by employing radioactive or fluorescent labels.

[0007] There are two general methods of forming polynucleotide arrays for the gene expression analysis. The first method involves in situ synthesis of oligonucleotides in predetermined positions of a solid substrate (Fodor et al, Science 251: 767-73, 1991). The second method includes the association of pre-synthesized oligonucleotides or polynucleotides with a solid substrate. In situ synthesis of oligonucleotides on glass modified with an aliphatic polyether linker (Southern, E. M. et al., Genomics 13:1008, 1992) and polypropylene as a solid substrate (U.S. Pat. No. 5,554,501) have been reported. The in situ method, however, suffers from certain shortcomings. For example, since the addition of each nucleotide is a separate reaction, the reproducibility and reaction yield may vary widely between different locations on the substrate, as well as between different substrates. Consequently, it is hard to obtain accurate and reproducible data with arrays formed by direct synthesis.

[0008] Alternatively, pre-synthesized polynucleotides may be immobilized on a substrate by ultraviolet cross-linking, chemical adhesion, or covalent bonding. Typically, a polynucleotide, a solid substrate, or both, are derivatized to initiate such immobilization. Usually it is preferred that the solid substrate is capable of immobilizing probe DNA, while being substantially inert to any other DNA. Glass is a commonly used solid substrate, because it is inexpensive and of good optical quality. Various types of pre-derivatized glass substrates are commercially available, including microscope slides coated with poly-L-lysine or amino propyl silane, or glass slides with exposed aldehyde functionalities. However, the derivatization of a glass surface creates a positive electrostatic net charge, which results in undesirable non-specific electrostatic binding of nucleic acids to the solid substrate during subsequent hybridization procedures. Numerous other surface coatings for the efficient immobilization of polynucleotides have been proposed and include an isolate of the naturally occurring mussel adhesive protein (U.S. Pat. No. 5,024,933), nucleoside phosphate (U.S. Pat. No. 4,818,681), and salt or cationic detergent (U.S. Pat. No. 5,610,287), to name a few. These methods, however, also entail unspecific binding of nucleic acids on the substrates. Such unspecific binding of DNA makes interpretation of the hybridization results difficult.

[0009] Polynucleotides themselves can be derivatized prior to the binding to a solid substrate. For example, U.S. Pat. No. 6,048,695 describes epoxide-modified DNA which is readily affixed to an unmodified solid substrate, such as an underivatized glass surface. Finally, both a polynucleotide and a solid substrate may be modified to allow efficient covalent bonding. For example, U.S. Pat. No. 5,215,882 discloses modifying a nucleic acid with a primary amine or equivalent, followed by the reaction of the modified nucleic acid with the solid substrate having free aldehyde groups in the presence of a reducing agent. However, the derivatization of polynucleotides and their subsequent covalent binding to solid substrates are long and tedious processes. Consequently, the arrays produced by conventional techniques are fairly expensive ($6-$36 per slide). Similar problems exist in respect to immobilization of other biomolecules on solid substrates.

[0010] In summary, the conventional immobilization methods do not provide desirable fast hybridization and high specificity of binding of targets to probes. Additionally, currently available methods of manufacturing assay articles for use in the detection of biomolecules are slow, tedious, and economically unfavorable.

SUMMARY OF THE INVENTION

[0011] Accordingly, it is an object of the invention to provide a cost-efficient, rapid and convenient method of making an assay article and a method of using such an assay article for the detection of target biomolecules. Particularly, it is an object of the present invention to develop a method of making an assay article by utilizing adsorption of biomolecules on a substrate.

[0012] The present invention is based on the discovery that modified plastic substrates, and particularly plasma aminated polypropylene substrates, are capable of stable immobilization of nucleic acids, proteins, polypeptides, and other biomolecules by adsorption. The inventors discovered that adsorption of biomolecules on modified substrates can be achieved by simply contacting biomolecules with the substrate and drying the substrate at ambient conditions. Although the immobilization of adhesive proteins on surfaces by adsorption is generally known (U.S. Pat. No. 5,024,933), the immobilization of biomolecules other than adhesive proteins by adsorption without chemical crosslinking or additional fixing steps, such as chemical treatment of the substrate and baking the substrate in an oven, has not been described. The present invention allows a stable immobilization of biomolecules other than adhesive proteins on substrates without chemical crosslinking and additional fixing steps.

[0013] Consequently, one aspect of the present invention provides a method of making an assay article for use in the detection of biomolecules. The method comprises the steps of:

[0014] (a) providing a first biomolecule and a second biomolecule, wherein the first biomolecule is a biomolecule other than an adhesive protein;

[0015] (b) providing a substrate having a modified surface;

[0016] (c) contacting the first biomolecule with the modified surface of the substrate and drying the substrate whereby the first biomolecule directly adsorbs and immobilizes on the modified surface of the substrate without additional fixing steps to form an activated substrate; and

[0017] (d) contacting the second biomolecule with the activated substrate under conditions sufficient for the first biomolecule to bind to or adsorb on the second biomolecule.

[0018] According to embodiments of the present invention, the substrate may be fabricated in a form of foams, filaments, threads, sheets, films, slides, gels, membranes, beads, plates, and like structures. In one embodiment of the present invention, the step of contacting the first biomolecule comprises:

[0019] (a) placing an aliquot of the first biomolecule solution on the substrate; and

[0020] (b) air-drying the substrate to directly adsorb the first biomolecule on the surface of the substrate.

[0021] In accordance with one embodiment of the present invention, a plurality of first biomolecules may be placed and adsorbed on the surface of the aminated polypropylene substrate in an array.

[0022] Another aspect of the present invention provides a method of detecting a target first biomolecule contained in a sample. The method comprises the steps of:

[0023] (a) providing an assay article of the present invention, wherein the second biomolecule can form a complex with the target biomolecule;

[0024] (b) contacting the assay article with the target biomolecule under a condition that allows the formation of a complex comprising the second biomolecule and the target biomolecule; and

[0025] (c) detecting and determining the presence of the complex as a measurement for the presence and/or the amount of the target biomolecule contained in the sample.

[0026] The complex of the second biomolecule and the target first biomolecules may also include a reporter. The reporter may be selected from a group consisting of: dyes, chemiluminescent compounds, enzymes, fluorescent compounds, metal complexes, magnetic particles, biotin, hapten, radio frequency transmitters, and radioluminescent compounds. The signal produced by the reporter molecules immobilized on the array may be detected and recorded by a number of means, such as a laser with a confocal array reader, phosphor imager, or a CCD camera.

[0027] A further aspect of the present invention provides an assay article for detecting biomolecules. The assay article comprises a substrate having a modified surface; a first biomolecule directly adsorbed and immobilized on the modified surface of the substrate without linking moieties; and a second biomolecule attached to the first biomolecule.

[0028] The present invention is well suited for use in creating biomolecular arrays, such as gene expression micro-arrays for use in gene expression analysis, in particular. The polynucleotide arrays may be used for the evaluation or identification of biological activity. Further, the assay articles of the present invention may contain a range of adsorbed first biomolecules to which a range of second biomolecules is bound and may be utilized in hybridization assays and immunoassays.

[0029] The present invention provides many advantages. Since the invention allows for the adsorption of first biomolecules directly on a solid substrate without chemical crosslinking, costly production of modified first biomolecules, such as thiol- or amino-modified DNA, may be avoided. Also, the task of making arrays is greatly simplified and the production costs are significantly reduced, because the first biomolecules are simply air-dried on the substrate. Furthermore, even if a particular biomolecule doesn't adsorb well on a particular substrate, it still may be attached to the substrate via first biomolecule. Thus, a broad range of assay devices with various biomolecules attached can be easily prepared in accordance with the present invention.

DESCRIPTION OF THE FIGURES

[0030] The above-mentioned and other features of this invention and the manner of obtaining them will become more apparent, and will be best understood by reference to the following description, taken in conjunction with the accompanying figures.

[0031]FIGS. 1A, 1B, 1C, and 1D schematically present methods of making assay devices according to embodiments of the present invention.

[0032]FIG. 2 shows the hybridization results of labeled target cDNA from actin, β-microglobulin, G3PDH, and p53 with their corresponding probe cDNA immobilized on polypropylene substrates. TNF-α immobilized on the substrate was used as a control for a non-specific hybridization.

[0033]FIG. 3 shows the attachment of human IgG to aminated polypropylene support.

DETAILED DESCRIPTION OF THE INVENTION

[0034] One aspect of the present invention provides a method of making an assay article for use in the detection of target biomolecules contained in a sample. As schematically shown in FIGS. 1A and 1B, the method comprises the steps of:

[0035] (a) providing a first biomolecule (2) and a second biomolecule (3, 9, 12, or 6), wherein the first biomolecule is a biomolecule other than an adhesive protein;

[0036] (b) providing a substrate (1) having a modified surface;

[0037] (c) contacting the first biomolecule (2) with the modified surface of the substrate and drying the substrate whereby the first biomolecule (2) directly adsorbs and immobilizes on the modified surface of the substrate without additional fixing steps to form an activated substrate; and

[0038] (d) contacting the second biomolecule (3, 9, 12, or 6) with the activated substrate under conditions sufficient for the first biomolecule (2) to bind or adsorb the second biomolecule (3, 9, 12, or 6).

[0039] The term “biomolecule,” as used herein, refers to nucleic acids, polypeptides, proteins, receptors, haptens, and analogues thereof. As used herein, “polynucleotide” refers to a polymer of deoxyribonucleotides or ribonucleotides, in the form of a separate fragment or as a component of a larger construction. “Polynucleotide”, as used herein, may be DNA, RNA, or a DNA analog, such as PNA (peptide nucleic acid). The DNA may be a single- or double-stranded DNA, or a DNA amplified by PCR technique. The RNA may be an mRNA. The length of the polynucleotides may be from about 20 bp to about 10 kb. In accordance with one embodiment of the present invention, the polynucleotide is a complementary DNA (cDNA). The length of a cDNA polynucleotide may be in the range of about 100 bp to about 10 kb, preferably, 200 bp to 1000 bp.

[0040] As used herein, “polypeptide” refers to a polymer of amino acids, wherein the α-carboxyl group of one amino acid is joined to the α-amino group of another amino acid by a peptide bond. A protein may comprise one or multiple polypeptides, linked together by disulfied bonds. Examples of the protein include, but are not limited to, antibodies, antigens, ligands, receptors, etc.

[0041] An antigen is a substance which is capable of inducing an immune response, i.e., antibody production, when introduced into an animal or human body. The region of an antigen that is recognized by an antibody and to which the antibody binds is referred to as an “epitope.” Although large molecules such as proteins or other “antigens” possess multiple epitopes, low molecular weight molecules, such as most pharmacological agents, possess only a single epitope. Such low molecular weight molecules are referred to herein as “haptens.”

[0042] It is a discovery of the present invention that first biomolecules may be attached to substrates by adsorption. As it is generally understood in the art, adsorption is a process in which atoms or molecules move from a bulk liquid phase onto a solid or liquid surface. Unlike the prior art that requires chemical cross-linking of biomolecules with the substrate (U.S. Pat. No. 6,322,968), fixing steps that immobilize adsorbed biomolecules on the substrate (U.S. Pat. No. 4,970,144), or the use of adhesive proteins (U.S. Pat. No 5,024,933), the present invention allows a stable immobilization of biomolecules (other than adhesive proteins) by direct adsorption on the substrate.

[0043] For the purposes of the present invention, the term “fixing step” refers to the immobilization of adsorbed biomolecules by baking the substrate in an oven at temperatures above 50° C. (U.S. Pat. No. 4,358,535) or treating the substrate with chemical agents, such as those used in conventional enzyme-linked immunosorbent assays (U.S. Pat. No. 4,970,144). For the purposes of the present invention, the term “adhesive protein” refers to polyphenolic proteins naturally occurring in mussels (U.S. Pat. No 5,024,933) and synthetic proteins having similar structural and adhesive properties. In the present invention, the term “direct adsorption” means adsorption by air-drying without the use of any chemical linkers and additional fixing steps as defined above.

[0044] Since the instant methods of assay article formation involve direct adsorption of first biomolecules on substrates, no chemical modification to first biomolecules is required. Both modified and unmodified first biomolecules may be immobilized on substrates by adsorption in accordance with the present invention. For the purpose of the present invention, “unmodified first biomolecule” means native first biomolecule, and “modified first biomolecule” means a first biomolecule with introduced functional groups. For example, a modified first biomolecule may be biotinylated DNA.

[0045] It has also been discovered by the inventors that polypropylene substrates are particularly useful for adsorption of biomolecules. The adsorption is further improved by using a substrate with a modified surface. For the purpose of the present invention, the term “modified surface” means a surface to which functional groups are added. In one embodiment, functional groups are selected from a group consisting of amino, carboxyl, thiol, hydroxyl and their derivatives. In one embodiment, the surface of the substrate is modified by the introduction of an amino group.

[0046] The methods for the introduction of amine groups onto the polypropylene surface are described in the commonly assigned U.S. Pat. No. 6,013,789, the relevant content of which is incorporated herein in its entirety by reference. In short, amino groups may be introduced onto the surface of a polypropylene medium by using a plasma discharge in an ammonia- or organic-amine-containing gas. The “plasma” is most preferably an ionized gas, which gains sufficient ionization energy from an electromagnetic field. Preferably, the ionization energy is applied by a radio-frequency plasma discharge, a microwave frequency plasma discharge, or a corona discharge. In a particularly preferred embodiment of the invention, the amine is derived from an ammonia gas and the elevated energy state is achieved via radio-frequency plasma discharge. The aminated polypropylene is then utilized for direct adsorption of a pre-synthesized first biomolecule.

[0047] In order to accommodate a number of different testing techniques including specialized testing equipment, aminated polypropylene substrates may be molded into any of a variety of shapes and forms. Examples of such shapes and forms of the aminated polypropylene substrates include, but are not limited to, foams, filaments, threads, sheets, films, slides, gels, membranes, beads, plates, and like structures. An aminated polypropylene substrate may be fabricated in the form of a planar device having discrete isolated areas in the form of wells, troughs, pedestals, hydrophobic or hydrophilic patches, die-cut adhesive reservoirs, or other physical barriers to fluid flow. Examples of such a substrate include, but are not limited to, a microplate or the like. Because the substrate of the present invention is particularly useful in the preparation of first biomolecule arrays for the evaluation or identification of biological activity, the aminated polypropylene substrate is preferably in the form of a device having at least one flat planar surface. Examples of such devices with flat surfaces include, but are not limited to, slides, sheets, films, or the like.

[0048] The size of the substrate can vary and depends upon the final use of the immobilized first biomolecules. Those skilled in the art will appreciate that arrays of first biomolecules immobilized on miniaturized solid supports have been under development for many years. These solid supports can be measured in terms of mm² and can have numerous different immobilized first biomolecules, each attached to a different site-specific location on the miniaturized solid support. Solid supports in the form of dipsticks and slides are also within the scope of the present invention. As known in the art, dipsticks typically are rectangular in shape with each side measuring a few centimeters.

[0049] In the present invention, a first biomolecule is immobilized on a substrate by contacting the first biomolecule with the modified surface of the substrate and drying the substrate. The immobilization of the first biomolecule on the modified surface of the substrate occurs without additional fixing steps. While not wanting to be bound by the theory, it is believed that, under the conditions of the present invention, first biomolecules may be adsorbed on the modified surface of the substrate by ionic and hydrophobic interaction. For the purposes of the present invention, the term “activated substrate” refers to a substrate with first biomolecules that are immobilized on its modified surface by adsorption.

[0050] For instance, in one embodiment, described in detail in the following Example 1, 10 nL aliquots of cDNA solutions are applied to an aminated polypropylene substrate. Following the application of the cDNA, the substrates are dried at 35° C. for 15 minutes. Then, the substrates are either soaked in ethanol for one hour or in ammonium hydroxide for 15 minutes to remove loosely bound nucleic acid. Finally, the slides are briefly rinsed with water and air-dried. One skilled in the art can readily determine the suitable conditions for adsorbing other first biomolecules in view of the teaching of the present invention. As discussed above, a variety of substrates may be used. In a preferred embodiment, however, aminated polypropylene substrates are used.

[0051] For the purpose of the present invention, it is not crucial which particular method is used to carry out the step of contacting the first biomolecule with the substrate. In accordance with embodiments of the present invention, the contacting step may be carried out by jet printing, solid or open capillary device contact printing, microfluidic channel printing, silk screening, and printing using devices based upon electrochemical or electromagnetic forces. For example, thermal inkjet printing techniques utilizing commercially available jet printers and piezoelectric microjet printing techniques, as described in U.S. Pat. No. 4,877,745, may be utilized to spot polynucleotides to the aminated substrates. A Biomek High Density Replicating Tool (HDRT) (Beckman Coulter, Calif.) may also be used for an automatic gridding. Alternatively, the contacting step may be carried out by manual spotting of the first biomolecules on the aminated substrate. Examples of manual spotting include, but are not limited to, manual spotting with a pipettor. It should be understood that the aminated substrate of the present invention may be exposed to first biomolecules by any methods as long as the first biomolecules are put in direct contact with the substrate.

[0052] In the present invention, the second biomolecules are contacted with the activated substrate under conditions sufficient for the first biomolecule to adsorb on or to bind to the second biomolecule. A condition is sufficient if it allows an interaction between the first and the second biomolecule resulting in the adsorption of the second biomolecule on the first biomolecule or the formation of a covalent or non-covalent bond between the second and the first molecules. Interaction between the first biomolecule and the second biomolecule include, but are not limited to, ionic, covalent, hydrophobic, Van der Waals, hybridization, and immuno reactions. The selection of such sufficient conditions is within the level of skill in the art and depends on the desired strength of the bond between the first and the second biomolecule. Such conditions include, but are not limited to, temperature, the ionic strength and viscosity of the buffer, and the respective concentrations of the first and the second biomolecules.

[0053] In one embodiment, the surface character (e.g., hydrophobicity, surface charge) of the first and the second biomolecules and the physio-chemical nature of the buffers and additives (e.g., pH, ionic strength, organic modifiers, surface blocking agents, etc.) used to prepare solutions of the first and the second biomolecules are selected or modified in such a way that the second biomolecule can adsorb on or to bind to the first biomolecule, which is immobilized on the surface of the substrate, while non-specific adsorption of the second biomolecule to the substrate is substantially avoided. For example, adsorption and/or binding between the first and the second biomolecules may be stimulated by providing the first biomolecules with a surface charge opposite to that of the second biomolecule to prevent electrostatic repulsion. The desirable surface charge may be obtained by an introduction of charged functional groups or by adjusting pH of buffers and additives used to prepare solutions of the first and the second biomolecules.

[0054] Another example of stimulating adsorption and/or binding between the first and the second biomolecules involves an introduction of functionalities of similar hydrophobicity onto the surface of the first and the second biomolecules to improve hydrophobic-hydrophobic or hydrophilic-hydrophilic interactions between the first and the second biomolecules. Other techniques are available for improving adsorption and/or binding between the first and the second biomolecules. Such techniques are well-known to those skilled in the art and will not be discussed here. An adsorption or binding of the second biomolecules to the first biomolecules advantageously permits an orientation of the second biomolecules toward the bulk solution phase for efficient capture of the analyte target.

[0055] As schematically shown in FIG. 1C, the first and the second biomolecules may be selected from a group consisting of specific binding partner pairs consisting of antigen (4) and antibody (3), hapten and antibody, hormone and receptor, nucleic acid strand (9) and complementary nucleic acid strand or complementary nucleic acid conjugate with a ligand (10) or antibody (13), and nucleic acid strand and sequence-specific nucleic acid binding protein. In one embodiment, the first biomolecule is a first polynucleotide and the second biomolecule is a second polynucleotide that is substantially complementary to the first polynucleotide. In this embodiment, the second polynucleotide that is “substantially complementary” is a known sequence of a nucleic acid that is designed to be sufficiently complementary to a sequence of the first nucleic acid, such that they will hybridize under selected stringency conditions.

[0056] In another embodiment, the first biomolecule is a first receptor or a first ligand and the second biomolecule comprises a second ligand or a second receptor, respectively, that recognizes and binds to the first receptor or ligand. In still another embodiment, the first biomolecule is an antigen or a hapten and the second biomolecule comprises an antibody that recognizes the antigen or the hapten, or vice versa.

[0057] In one embodiment, the first biomolecule is a protein and the second biomolecule is a nucleic acid or an antibody. For example, the first biomolecule may be albumin and the second biomolecule may be an antibody for albumin or a nucleic acid conjugated to albumin.

[0058] Methods of conjugating biomolecules are generally known to those skilled in the art and will be discussed here only briefly. For example, a number of strategies are known for covalent coupling of nucleic acid molecules to proteins. Ultraviolet cross-linking can effectively immobilize DNA molecules to proteins (Jang, et al., J. Immuno. 145: 3353-3359 (1990)). Chemical cross-linking also can be very efficient. One of these methods is coupling 5′ Thiol oligos to sulfo-GMBS activated and β-mercaptoethanol treated proteins (Schweitzer, et al., Proc. Natl. Acad. Sci. USA, 97:10113-10119 (2000)).

[0059] Another method is coupling 5′ amine-modified oligos to proteins through an amine crosslinker reagent. Glutaraldehyde can also be used to couple 5′ amine-modified oligos to proteins. In this case, the 5′ amine-reference primer is incubated with 2% glutaraldehyde. After ethanol precipitation or column purification is carried out to remove the unused glutaraldehyde, the primer-glutaraldehyde complex is incubated with proteins, resulting in the following structure: reference primer-N═CH—(CH₂)₃—CH═N-protein. The oligonucleotide conjugated proteins are finally subjected to gel filtration purification to remove free oligonucleotides. Coupling DNA primers to other types of probe molecules is also possible. Chemical reactions depend on the nature of the probe molecules.

[0060] In one embodiment, the second biomolecule is conjugated to a receptor. The receptor may be an antigen or an antibody.

[0061] In accordance with embodiments of the present invention, the step of providing the first biomolecule may include providing a solution of the first biomolecule. The step of contacting the first biomolecule with aminated substrate may include:

[0062] (a) placing an aliquot of the first biomolecule solution on the modified surface of the substrate; and

[0063] (b) air-drying the substrate to directly adsorb the first biomolecule on the modified surface of the substrate.

[0064] The solution of the first biomolecule may be any solution that delivers the first biomolecule to the surface of the substrate. Preferably, the solvent is an aqueous buffer having a pH from about 4 to about 13. In one embodiment, the first biomolecule is a polynucleotide, and a solution of the polynucleotides in 50 mM sodium bicarbonate, pH 9, is spotted on the aminated substrate.

[0065] The concentration of first biomolecules contained in aqueous solutions may vary, depending on the type of molecule, the molecule size, the molecule structure, and other factors that may influence the solubility of the molecules. Preferably, the amount of the first biomolecules applied to the substrate ranges from about 10⁻²⁰ to about 10⁻¹⁴ moles. For example, in one embodiment, the first biomolecule is a polynucleotide, and the amount of the polynucleotide applied to the substrate is about 10⁻¹⁸ moles. The size of the first biomolecule solution aliquot is not crucial, as long as it provides a sufficient amount of the first biomolecule. Consequently, the size of the aliquots applied to the aminated substrate may vary, depending on the concentration of the first biomolecule in the solution and the assay needs. For example, the aliquot may be from about 0.1 nL to about 500 nL. In one embodiment, the first biomolecule is a polynucleotide, and aliquots of about 10 nL of the 1 nM polynucleotide solutions are placed on the aminated substrate.

[0066] In accordance with the present invention, the air-drying step is conducted for a period of time sufficient to allow adsorption of the first biomolecule solution. The length of the air-drying time depends on the volume of the aliquots applied to the substrate, room temperature and humidity. For micro- and nanoliter aliquots, the air-drying step may take from about 5 to about 60 minutes. For example, in one embodiment, 10 nL aliquots are placed on the surface of the aminated substrate and dried at 22° C. for one hour or for about fifteen minutes at 35° C.

[0067] As mentioned above, many applications for utilizing immobilized biomolecules require biomolecules to be immobilized at site-specific locations on a substrate surface. Accordingly, in the present invention, a plurality of first biomolecules may be placed and adsorbed on the surface of the aminated polypropylene substrate in an array. In order to prepare ordered arrays of first biomolecules with each first biomolecule located at site-specific locations, including grids and 1×n arrays of immobilized first biomolecules, a preselected site on the surface of the substrate is exposed to a solution of the desired first biomolecule. In accordance with the present invention, this can be accomplished manually by applying an aliquot of first biomolecule solution to a preselected location on the substrate. Alternatively, thermal inkjet printing techniques utilizing commercially available jet printers and piezoelectric microjet printing techniques, as described in U.S. Pat. No. 4,877,745, can be utilized to spot-selected substrate surface sites with selected first biomolecules.

[0068] A wide variety of array formats may be employed in accordance with the present invention. One particularly useful format is a linear array of nucleic acid probes, generally referred to in the art as a dipstick. Another suitable format comprises a two-dimensional pattern of discrete cells. Of course, as would be readily appreciated by those skilled in the art, other array formats would be equally suitable for use in accordance with the present invention.

[0069] The array of the present invention may be a part of a variety of devices, such as microtiter plates, test tubes, inorganic sheets, dipsticks, etc. For example, when the substrate is a thread, one or more of such threads can be affixed to a plastic dipstick-type device. When the substrate is in a form of a membrane, it can be affixed to glass slides. The particular device is, in and of itself, unimportant, as long as the substrate is securely affixed to the device without affecting the functional behavior of the substrate or any adsorbed first biomolecule. The device should also be stable to any materials into which the device is introduced (e.g., clinical samples, etc.).

[0070] The utility of the present invention is further extended by binding the second biomolecules to the first biomolecules that are immobilized on a substrate by adsorption. Even if a particular biomolecule doesn't adsorb well on a particular substrate, it still may be attached to the substrate via first biomolecule, which serves as a bridge between the surface and the second biomolecule. Thus, a broad range of assay devices with various biomolecules attached can be easily prepared in accordance with the present invention. In a preferred embodiment, the first biomolecules are immobilized by adsorption at site-specific locations on a substrate surface in an array format. The second biomolecules are adsorbed on or bound to the first biomolecules to form an array of the second biomolecules.

[0071] In one embodiment, after immobilization of the first biomolecules, the modified surface of the substrate is masked or chemically treated to prevent unspecific binding of the second biomolecules to the modified surface of the substrate. In another embodiment, the surface character (hydrophobicity, surface charge, polymer composition), the physio-chemical nature of the first and the second biomolecules (size, shape, charge, and hydrophobicity), and the physio-chemical nature of buffers and additives used to prepare solutions of the first and the second biomolecules are selected or modified in such a way that the first biomolecule can adsorb on the surface of the substrate, while non-specific adsorption of the second biomolecule to the substrate is substantially avoided. An adsorption or binding of the second biomolecules to the first biomolecules advantageously permits an orientation of the second biomolecules toward the bulk solution phase for efficient capture of the analyte target.

[0072] As schematically shown in FIG. 1D, in still another embodiment, the second biomolecule (9) is conjugated to the first biomolecule (2) to form a conjugate (16). The second biomolecule (9) itself can be bound or conjugated to a third biomolecule (15) or a conjugate (13) to form a conjugate (14). Then, the conjugate (16) is contacted with the modified surface of the substrate and the substrate is dried. As a result, the first biomolecule becomes directly adsorbed and immobilized on the modified surface of the substrate, while the second biomolecule stays exposed for binding with a target biomolecule. In this embodiment, the surface properties, the nature of the first and the second biomolecules, and other adsorption conditions are such that adsorption of the first biomolecule on the substrate surface is greatly favored over the adsorption of the second biomolecule. For example, when conjugate is a protein-nucleic acid complex, such as albumin-nucleic acid conjugate, the substrate surface, such as surface of aminated polypropylene, may favor adsorption of the protein due to to hydrophobic-hydrophobic interactions.

[0073] The method of making an assay article may further include a step of exposing the assay article with immobilized first and second biomolecules to a reagent for removing loosely bound first and second biomolecules. Examples of such reagents include, but are not limited to, ammonium hydroxide, ethanol, and protein. In a preferred embodiment of this invention, ethanol is used for nucleic acid arrays and casein is used for protein arrays.

[0074] Another aspect of the present invention provides a method of detecting a target biomolecule contained in a sample. As schematically shown in FIG. 1A, the method comprises the steps of:

[0075] (a) providing the assay article of the present invention, wherein the second biomolecule (3 or 9) can form a complex with the target biomolecule (4 or 10);

[0076] (b) contacting the assay article with the target biomolecule (4, 10) under a condition that allows the formation of a complex comprising the second biomolecule and the target biomolecule; and

[0077] (c) detecting and determining the presence of the complex as a measurement for the presence or the amount of the target biomolecule contained in the sample.

[0078] For the purpose of the present invention, the second biomolecule (probe) recognizes and binds to the target biomolecule forming a complex. The target and second biomolecules may be selected from a group consisting of nucleic acids, polypeptides, proteins, and their analogues. For example, when the target is a polynucleotide, the probe may comprise a polynucleotide that is complementary to the target polynucleotide (see FIG. 1). When the target is a receptor or a ligand, the probe may comprise a ligand or a receptor that respectively recognizes and binds to the target receptor or ligand. When the target is an antigen, the probe may comprise an antibody that recognizes the antigen, or vice versa (see FIG. 2).

[0079] Contacting the probes with the targets (or hybridization) is conducted under conditions that allow the formation of stable complexes between probes and targets. For example, when target polynucleotides are contacted with probe polynucleotides (second biomolecules) bound to the first biomolecules adsorbed on an aminated polypropylene substrate, complementary regions on the target and the probe polynucleotides anneal to each other, forming a probe-target (second biomolecule-target) complex. The selection of such conditions is within the level of skill in the art and include those in which a low, substantially zero, percentage of mismatched hybrids form. The precise conditions depend, however, on the desired selectivity and sensitivity of the assay. Such conditions include, but are not limited to, the hybridization temperature, the ionic strength and viscosity of the buffer, and the respective concentrations of the target and probe biomolecules. Hybridization conditions may be initially chosen to correspond to those known to be suitable in standard procedures for hybridization to filters and then optimized for use with the aminated polypropylene substrates of the present invention. The conditions suitable for the hybridization of one type of target material would appropriately be adjusted for use with other target materials.

[0080] For example, in certain embodiments, the target polynucleotides are hybridized to the probe polynucleotides at temperatures in the range of about 20° C. to about 70° C., for a period from about 1 hour to about 24 hours, in a suitable hybridization buffer. Suitable hybridization buffers for use in the practice of the present invention generally contain a high concentration of salt. A typical hybridization buffer contains in the range of about 2× to about 6×SSC and about 0.01% to about 0.5% SDS at pH 7-8. Once the probe/target complex is formed, the substrates are washed under conditions suitable to remove substantially all non-specifically bound target or probe biomolecules. Preferably, the washing is carried out at a temperature in the range of about 20° C.-70° C. with a buffer containing about 0.1-6×SSC and 0.01-0.1% SDS. The most preferred wash conditions for polynucleotides presently include a temperature, which is the same as hybridization temperature, and a buffer containing 2×SSC and 0.01% SDS. As previously noted, it would be a routine matter for those working in the field to optimize the contacting (hybridization) conditions for any given combination of target and probe biomolecules.

[0081] In accordance with embodiments of the present invention, either the targets or probes may be labeled with a reporter. Detectability may be provided by such characteristics as color change, luminescence, fluorescence, or radioactivity. Examples of reporters include, but are not limited to, dyes, chemiluminescent compounds, enzymes, fluorescent compounds, metal complexes, magnetic particles, biotin, haptens, radio frequency transmitters, and radioluminescent compounds. One skilled in the art can readily determine the type of reporter to be used once the type of target or probe biomolecules is determined.

[0082] The labeling procedure may occur prior to analysis (direct labeling) or after hybridization (indirect labeling). An example of indirect labeling would be the biotinylation of a target polynucleotide, hybridizing it with a probe, and reacting the target-probe complexes with a streptavidin-alkaline phosphatase conjugate. The biotin moieties retained after the hybridization with probe polynucleotides bind to a streptavidin-alkaline phosphatase conjugate, which then acts on a chromogenic substrate, such as Enzyme Labeled Fluorescent (ELF) reagent.

[0083] For the purpose of the present invention, the same or different first and second biomolecules may be attached to the substrates. If the first and/or second biomolecules are different, preferably they are located in isolated areas of the substrate to form arrays. For example, a substrate may be a microplate. Different first biomolecules may be adsorbed within different wells of the microplate for forming arrays. Then, different second biomolecules may be bound to the corresponding first biomolecules. In accordance with another embodiment of the present invention, the substrate may be a slide and different first biomolecules may be adsorbed on different areas of the slide to form an array.

[0084] The signal produced by an array may be detected by a naked eye or by means of a specially designed instrumentation, such as a confocal array reader. For example, in one embodiment, a fluorescent signal is recorded with a charge coupled device (CCD) camera. It would be appreciated by those skilled in the art that the choice of a particular method used to detect and quantify the signal is not crucial for this invention. Essentially, any detection method may be used as long as it provides consistent and accurate results.

[0085] Another aspect of the present invention provides an assay article for detecting target biomolecules. The assay article of the present invention comprises a substrate having a modified surface; a first biomolecule directly adsorbed and immobilized on the modified surface of the substrate without linking moieties; and a second biomolecule attached to the first biomolecule.

[0086] The substrate may be made of a variety of materials. In one embodiment, the substrate is made of polypropylene. Polypropylene is organic material that can be surface activated, but otherwise is chemically inert under harsh chemical conditions. Polypropylene can be used in very corrosive environments. For example, polypropylene has good chemical resistance to a variety of mineral acids (e.g., hydrochloric acid), organic acids (e.g., formic acid, acetic acid), bases (e.g., ammonium hydroxide, potassium hydroxide), salts (e.g., sodium chloride), oxidizing agents (e.g., peracetic acid, iodine solutions), and organic solvents (e.g. acetone, ethyl alcohol, acetonitrile, dichloromethane, etc.). Additionally, polypropylene is hydrophobic and provides a low fluorescence background. Amino groups may be introduced onto the polypropylene surface by using a plasma discharge in an ammonia or organic-amine-containing gas, as described above.

[0087] The assay article of the present invention may be molded into a variety of shapes, including, but not limited to, foams, filaments, threads, sheets, films, slides, gels, membranes, beads, plates, and like structures.

[0088] The assay articles of the present invention are well suited for use as genosensors and other array-based systems, such as differential gene expression micro-arrays. The assay articles of the present invention may also be used as devices for performing a ligand binding assay or for performing a hybridization assay by either reverse hybridization (probes attached) or Southern dot blot (target attached). The assay articles of the present invention may also be used in immunoassays.

[0089] The invention may be better understood with reference to the accompanying examples that are intended for purposes of illustration only and should not be construed, in any sense, as limiting the scope of the present invention, as defined in the claims appended hereto. While the described procedures in the following examples are typical of those that might be used, other procedures known to those skilled in the art may alternatively be utilized. Indeed, those of ordinary skill in the art can readily envision and produce further embodiments, based on the teachings herein, without undue experimentation.

Example 1

[0090] Preparation of cDNA Arrays

[0091] Polypropylene slides were surface aminated by a radio-frequency discharge into ammonia gas, as described in Coassin et al. (U.S. Pat. No. 5,554,501, assigned to the assignee of the present invention). Unmodified cDNA of actin, β-microglobulin, G3PDH, p53, and TNF-A genes were prepared, each at a final concentration of 1 nM in 50 mM sodium bicarbonate buffer, pH 9. A Biomek 2000™ robotic system (Beckman Coulter, Inc., CA) equipped with a 384-pin HDRT system (Beckman Coulter, Inc., CA) was used to apply 10 nL aliquots of cDNA solutions onto the aminated polypropylene slides. A set of 5-(Biotinamido)pentylamine (BAPA) markers (Pierce Chemical, IL) were also printed at both ends of each slide, thereby flanking the cDNAs. BAPA, which binds streptavidin-enzyme conjugate independently of hybridization, serves as an internal control for assay robustness. Following the application of the cDNA and BAPA markers, the slides were dried at 35° C. for 15 minutes. Then the slides were either soaked in ethanol for one hour or in ammonium hydroxide for 15 minutes to remove loosely bound nucleic acid. Then the slides were briefly rinsed with water and air-dried. In one case, an ethanol-quenched slide was further rinsed in 1M NaOH for 15 minutes. The slides were stored at −20° C. until needed for experiments.

[0092] Hybridization

[0093] For hybridization, each slide was brought to room temperature and denatured for 15 minutes in 0.1 5M NaCl and 0.5M NaOH solution. A mixture of biotin-labeled cDNA targets of actin, β-microglobulin, G3PDH, and p53 were applied at a final concentration of 0.5 nM, following denaturation and the addition of hybridization buffer (2.4×SSC, 0.016% SDS, 0.28M TRIS, 0.028 NaCl, pH 7.5). The TNF-α target was not added so that non-specific hybridization could be measured to the TNF-α probe. Hybridization was allowed to proceed for 1 hour at 60° C., followed by a stringency rinse in 2×SSC, 0.01% SDS at the same temperature. The slides were incubated with streptavidin-alkaline phosphatase, and ELF reagent (fluorescent substrate for alkaline phosphatase) for 30 min. Following the incubation, the fluorescent signal image was recorded using a CCD camera.

[0094] Results

[0095] An array of cDNA and BAPA markers were successfully adsorbed on the aminated polypropylene slides, as determined by the hybridization of a mixture of the corresponding cDNAs (FIG. 2). FIG. 2 shows Biomek HDRT printing of cDNA and BAPA markers onto aminated polypropylene slides. The TNF-α signal was absent (negative control), while the remaining cDNA hybridization signals remained approximately at the same intensity as the signals from the BAPA markers (positive control). The signal from the ammonium-hydroxide-quenched slide was significantly reduced in the intensity over that of the ethanol-quenched slides. There was no significant difference between ethanol- vs. ethanol-NaOH-pretreated slides. This example illustrates that plasma aminated polypropylene slides are capable of direct and stable adsorption of cDNA without chemical crosslinking.

[0096] Thus, the aminated polypropylene slides with directly adsorbed first biomolecules of the present invention and the method of their use in detection of target biomolecules are well adapted to attain all of the ends and objects set forth above, together with other advantages, which are inherent to the system. The present invention may be embodied in other specific forms without departing from its essential characteristics. The described embodiment is to be considered in all respects only as illustrative and not as restrictive. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description. All changes which come within the meaning and range of the equivalence of the claims are to be embraced within their scope.

EXAMPLE 2

[0097] Preparation of Protein Array

[0098] Human IgG attachment: Polypropylene film was surface aminated by a radio-frequency discharge as described in Example 1. Diluted Human IgG (Pierce Chemicals, cat. # 31877, 28 mg/mL) stock to 1 mg/mL in sodium bicarbonate (50 mM, pH 9) and 4% sodium sulfate. Twenty-one 0.5 μL spots were pipetted onto an amino polypropylene strip 2 cm wide and 8 cm long. Attachment reaction was allowed to proceed for 60 min. at 25° C. The film was rinsed with Casein solution (1 mg/mL in 50 mM sodium carbonate, 0.1 5M NaCl pH 9) for 60 min. at 25° C., and then rinsed twice in deionized water and by 1× TBS, 0.02% Tween-20, pH 7.4 briefly. The strip was then used for binding assay.

[0099] Conjugation with goat anti-human IgG alkaline phosphatase: The 200 μL of diluted goat anti-human IgG alkaline phosphatase (Pierce Chemicals, cat. # 31310) solution 1:1000 in blocking buffer (1×TBS, 1 mg/mL Casein, 0.02% Tween-20 pH 7.4) was pipetted to a petri dish and the above-spotted polypropylene strip was placed spotted side down on top of the solution and was incubated for 60 min. at 25° C. The polypropylene strip was then rinsed 4 times in 20 mL of 1×TBS, 0.02% Tween-20, pH 7.4.

[0100] ELF detection: To detect the fluorescence signal, the enzyme substrate, ELF, was prepared by mixing components A and B (1:25) (Molecular Probes, Eugene, Oreg.) and 200 μL solution for each strip was used as described above. After 30 min. incubation at 22° C., the strip was dipped once in deionized water. The signals were detected using 365 nm UV light and a CCD camera, having 520 nm filter as shown in FIG. 3.

[0101] Results

[0102] An array of IgG was successfully adsorbed on the aminated polypropylene film as shown in FIG. 3.

[0103] Thus, the aminated polypropylene slides with directly adsorbed first biomolecules of the present invention and the method of their use in detection of target first biomolecules are well adapted to attain all of the ends and objects set forth above, together with other advantages, which are inherent to the system. The present invention may be embodied in other specific forms without departing from its essential characteristics. The described embodiment is to be considered in all respects only as illustrative and not as restrictive. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description. All changes which come within the meaning and range of the equivalence of the claims are to be embraced within their scope. 

What is claimed is:
 1. A method of making an assay article for use in detection of a target biomolecule comprising the steps of: (a) providing a first biomolecule and a second biomolecule, wherein the first biomolecule is a biomolecule other than an adhesive protein; (b) providing a substrate having a modified surface; (c) contacting the first biomolecule with the modified surface of the substrate and drying the substrate whereby the first biomolecule directly adsorbs and immobilizes on the modified surface of the substrate without additional fixing steps to form an activated substrate; and (d) contacting the second biomolecule with the activated substrate under conditions sufficient for the first biomolecule to adsorb on or bind to the second biomolecule.
 2. The method of claim 1, wherein the substrate is in a form selected from the group consisting of foams, filaments, threads, sheets, films, slides, gels, membranes, beads, plates, and like structures.
 3. The method of claim 1, wherein the contacting step (c) is carried out by a technique selected from a group consisting of jet printing, solid or open capillary device contact printing, microfluidic channel printing, silk screening, printing using devices based upon electrochemical or electromagnetic forces, and manual spotting.
 4. The method of claim 1, wherein the first biomolecule and the second biomolecule are selected from a group consisting of nucleic acids, polypeptides, proteins, receptors, haptens, and analogues thereof.
 5. The method of claim 1, wherein the first biomolecule and the second biomolecule are selected from a group of specific binding partner pairs consisting of antigen and antibody, hapten and antibody, hormone and receptor, nucleic acid strand and complementary nucleic acid strand, and nucleic acid strand and sequence-specific nucleic acid binding protein.
 6. The method of claim 5, wherein the first biomolecule is nucleic acid strand and the second biomolecule is a complementary nucleic acid strand.
 7. The method of claim 6, wherein the second biomolecule is conjugated to a receptor.
 8. The method of claim 7, wherein the receptor is selected from a group consisting of antigens and antibodies.
 9. The method of claim 1, wherein the second biomolecule is conjugated to the first biomolecule to form a conjugate, and wherein the contacting steps (c) and (d) comprise contacting the conjugate with the modified surface of the substrate and drying the substrate whereby the first biomolecule directly adsorbs and immobilizes on the modified surface of the substrate without additional fixing steps.
 10. The method of claim 9, wherein the first biomolecule and the second molecule are independently selected from a group consisting of nucleic acids, polypeptides, proteins, receptors, haptens, and analogues thereof.
 11. The method of claim 10, wherein the first biomolecule is a protein.
 12. The method of claim 11, wherein the second biomolecule is selected from a group consisting of nucleic acids and antibodies.
 13. The method of claim 12, wherein the first biomolecule is albumin and the second biomolecule is an antibody for albumin.
 14. The method of claim 13, wherein the antibody is conjugated to a third biomolecule selected from a group consisting of nucleic acids, polypeptides, proteins, receptors, haptens, and analogues thereof.
 15. The method of claim 12, wherein the first biomolecule is albumin and the second biomolecule is a nucleic acid.
 16. The method of claim 1, wherein the step of providing the first biomolecule comprises providing a solution of the first biomolecule; and the contacting step (c) comprises: (a) placing an aliquot of the first biomolecule solution on the modified surface of the substrate; and (b) air-drying the substrate at ambient temperature to directly adsorb the first biomolecule on the modified surface of the substrate.
 17. The method of claim 16, wherein the amount of the first biomolecule applied to the substrate ranges from about 10⁻²⁰ to about 10⁻¹⁴ moles.
 18. The method of claim 17, wherein the aliquot is from about 0.1 nL to about 500 nL.
 19. The method of claim 16, wherein the air-drying step is conducted for a period ranging from about 5 minutes to about 60 minutes.
 20. The method of claim 1, wherein the substrate is made of plastic.
 21. The method of claim 1, wherein a plurality of the first biomolecules are placed and adsorbed on the surface of the plastic substrate in an array.
 22. The method of claim 20, wherein the plastic is polypropylene.
 23. The method of claim 1, wherein the step of (b) further comprises a step of modifying the surface of the substrate to stimulate adsorption of the first biomolecules, while substantially preventing non-specific adsorption of the second biomolecules.
 24. The method of claim 23, wherein the modifying comprises introduction of a functionality selected from a group consisting of amino, carboxyl, hydroxyl, thiol, and their derivatives.
 25. The method of claim 24 wherein the functionality is an amino group.
 26. A method of detecting a target biomolecule contained in a sample comprising the steps of: (a) providing an assay article of claim 1, wherein the second biomolecule can form a complex with the target biomolecule; (b) contacting the assay article with the target biomolecule under a condition that allows the formation of a complex comprising the second biomolecule and the target biomolecule; and (c) detecting and determining the presence of the complex as a measurement for the presence or the amount of the target biomolecule contained in the sample.
 27. The method of claim 26, wherein the complex further comprises a reporter selected from the group consisting of dyes, chemiluminescent compounds, enzymes, fluorescent compounds, metal complexes, magnetic particles, biotin, haptens, radio frequency transmitters, and radioluminescent compounds.
 28. An assay article prepared in accordance with the method of claim
 1. 29. An assay article comprising: a substrate having a modified surface; a first biomolecule directly adsorbed and immobilized on the modified surface of the substrate without linking moieties; and a second biomolecule attached to the first biomolecule.
 30. The assay article of claim 29, wherein the second biomolecule is chemically bound to the first biomolecule. 31 The assay article of claim 29, wherein the second biomolecule is adsorbed to the first biomolecule.
 32. The assay article of claim 29, wherein the second biomolecule is conjugated to a third biomolecule.
 33. The assay article of claim 29, wherein the substrate is in a form selected from the group consisting of foams, filaments, threads, sheets, films, slides, gels, membranes, beads, plates, and planar devices having discrete isolated areas in the form of wells, troughs, pedestals, hydrophobic or hydrophilic patches, die-cut adhesive reservoirs, or other physical barriers to fluid flow.
 34. The assay article of claim 29, wherein the first biomolecule and the second biomolecule are selected from a group of specific binding partner pairs consisting of antigen and antibody, hapten and antibody, hormone and receptor, nucleic acid strand and complementary nucleic acid strand, and nucleic acid strand and sequence-specific nucleic acid binding protein. 